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Biomedical subjects

D W Lincoln

Publications and source records attributed to D W Lincoln.

At least 19 recordsLinked to original sources
PubMed1992

17 beta-estradiol hydroxylation catalyzed by human cytochrome P450 1A1: a comparison of the activities induced by 2,3,7,8-tetrachlorodibenzo-p-dioxin in MCF-7 cells with those from heterologous expression of the cDNA.

Exposure of MCF-7 breast cancer cells to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) causes an elevated cytochrome P450 content and a marked increase in the microsomal hydroxylation of 17 beta-estradiol (E2) at the C-2, C-4, C-15 alpha, and C-6 alpha positions. In this study we investigated the involvement of cytochromes P450 of the 1A gene subfamily in this metabolism of E2. Hydroxylation at each of these four positions of E2 was inhibited by P450 1A-subfamily inhibitors, alpha-naphthoflavone, benzo[a]pyrene, and 7-ethoxyresorufin. Northern blots showed that treatment of MCF-7 cells with TCDD resulted in production of the 2.6-kb CYP1A1 mRNA, but not the 3.0-kb CYP1A2 mRNA. Immunoblot analyses with anti-P450 1A antibodies confirmed the production of P450 1A1 protein in TCDD-treated MCF-7 cells. Anti-rat P450 1A IgG inhibited the hydroxylation of E2 at C-2, C-15 alpha, and C-6 alpha, but not hydroxylation at C-4. E2 hydroxylation by human cytochromes P450 1A1 and P450 1A2 was assessed in experiments with microsomes from Saccharomyces cerevisiae after transformation with cDNAs encoding the two cytochromes. The major hydroxylase activities of expressed human P450 1A1 were at the C-2, C-15 alpha, and C-6 alpha positions of E2; expressed human P450 1A2 catalyzed hydroxylation predominately at C-2. While both expressed P450s 1A1 and 1A2 had minor hydroxylase activities at the C-4 position, neither catalyzed a low-Km hydroxylation at C-4 similar to that observed with microsomes from TCDD-treated MCF-7 cells. These results provide strong evidence that P450 1A1 catalyzes the hydroxylations of E2 at the C-2, C-15 alpha, and C-6 alpha in incubations with microsomes from TCDD-treated MCF-7 cells, but suggest TCDD may also induce a cytochrome P450 E2 4-hydroxylase that is distinct from P450 1A1 or P450 1A2.

Breast Neoplasms↗
PubMed1992

Human contraception: development of new scientific opportunities.

Our current methods of contraception were largely developed in the 1950s and 1960s. Collectively these methods can limit population growth, although their acceptability at a personal level remains marginal. The number of abortions in the UK has risen progressively, from < 130,000 in 1980 to > 170,000 in 1990, and more people have sought sterilization. In global terms, population continues to increase on a scale unrecorded in human history. More than 800 million people, equal to the current population of North America, Europe and Japan, will be added to the world population of 5400 million by the year 2000, with 95% of the increase located in the economically and environmentally fragile regions of the South. Worldwide, in these 8 years, about 500 million abortions will be performed (> 40% by unsafe methods), about 80 million children under 1 year of age will die, and about 5 million women with be lost from pregnancy-related causes. Over 100 million people, most in Africa, Latin America, India and South-east Asia, will have become infected with human immunodeficiency virus. Religious, commercial and political pressures continue to constrain the development and distribution of contraceptive products, though some changes are in sight. Contraception is now being advanced in the broader context of neonatal and maternal health care. New scientific opportunities are also evident, with the potential to advance contraception in a quantal step. Third generation contraceptive steroids, anti-steroids, steroid-releasing vaginal rings, and injectable steroid preparations for men are all under clinical trial. Developments in the longer term, however, could hinge upon the greater specificity afforded by the manipulation of regulatory peptides. Agonists, antagonists and binding proteins for GnRH and the gonadotrophins are being investigated in an attempt to regulate the pituitary-gonadal axis more precisely. Inhibition of this axis may require the provision of low level steroid replacement, appropriately titrated to provide positive health care in the context of menstrual cyclicity, well-being, breast cancer and osteoporosis. Attempts are being made to identify and intercept the highly specific signals involved in fertilization and the maternal recognition of pregnancy, with the current clinical focus on the immunoneutralization of beta human chorionic gonadotrophin (beta hCG). More specific and more acceptable targets might include peptides involved in sperm-zona adhesion and sperm activation, sperm-oocyte fusion, and sperm-activated oocyte cleavage. The interception of these signals would prevent fertilization, implantation or both, and yet leave the hormonal profile of the menstrual cycle unchanged.(ABSTRACT TRUNCATED AT 400 WORDS)

Contraception↗
PubMed1991

Estrogen-stimulation of postconfluent cell accumulation and foci formation of human MCF-7 breast cancer cells.

Foci, nodules of cellular overgrowth, that appear after confluence are an in vitro characteristic of malignant transformation. A well-studied in vitro model of estrogen-dependent tumors is the MCF-7 cell line, derived from a pleural metastasis of a human breast adenocarcinoma. We report that cultivation of MCF-7 cells, using routine methods, results in extensive estrogen-stimulated postconfluent cell accumulation characterized by discrete three-dimensional arrays. Side view Nomarski optical sections revealed these to be principally multicellular foci with occasional domes and pseudoacinar vacuoles. This effect on MCF-7 cell growth occurs in media containing fetal bovine serum but not with calf serum or charcoal-dextran-treated fetal bovine serum unless supplemented with estrogens. Foci formation starts 5-6 days after confluence, and the number of foci generated is a function of the concentration of added estrogens. Foci formation is suppressed by the antiestrogens Tamoxifen and LY 156758. Addition of progesterone, testosterone, or dexamethasone had little or no effect, while various estrogens (ethinyl estradiol, diethylstilbestrol, and moxestrol) induced foci development. Clones derived from single cells of the initial MCF-7 population revealed a wide variance in estrogen-induced foci formation, demonstrating heterogeneity of this tumor cell line. The postconfluent cell growth of the estrogen receptor-deficient cell line, MDA-MB-231, contrasted with MCF-7 by developing an extensive multilayer morphology devoid of discrete structures. The tumorigenic potential of the MCF-7 cells used in our experiments was confirmed by their estrogen-dependent growth in immunosuppressed male BDF1 mice. These data suggest an estrogen receptor-based mechanism for the development of multicellular foci during postconfluent growth of MCF-7 cells. After confluence, foci, in contrast to the quiescent surrounding monolayer, retain proliferating cells. Focus formation, therefore, reflects the heterogeneous responsiveness of these cells to estrogens and should provide a model permitting in vitro comparisons between the progenitor cells of multicellular foci and the monolayer population.

Adenocarcinoma↗
PubMed1990

2,3,7,8-Tetrachlorodibenzo-p-dioxin causes an extensive alteration of 17 beta-estradiol metabolism in MCF-7 breast tumor cells.

MCF-7 breast tumor cells form multicellular foci in vitro when supplemented with 17 beta-estradiol (E2). In the presence of E2 and the aryl hydrocarbon-receptor agonist 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), MCF-7 cells grow to confluence but do not form foci. To investigate the role of E2 metabolism in this antiestrogenic effect of TCDD, analyses were performed by capillary GC/MS. The results revealed that pretreatment of MCF-7 cultures with TCDD (10 nM) rapidly depletes E2. In untreated cultures supplemented with 10 nM E2, the concentration of free E2 decreased to 4 nM in the first 12 hr, followed by a slower rate of decline. After 3 days most E2 in the medium was in conjugated form(s); 1.7 nM was present as free E2, and 2.9 nM was released by treatment with glucuronidase/sulfatase. In TCDD-treated cultures, E2 declined to 290 pM in 12 hr and after 2 days was not detected (less than 100 pM) either as free steroid or after treatment with glucuronidase/sulfatase. Intracellular E2 and estrone were likewise depleted by pretreatment with TCDD. Microsomes from TCDD-treated cells showed highly elevated aryl hydrocarbon-hydroxylase activity and catalyzed hydroxylations of E2 at C-2, C-4, C-15 alpha, and C-6 alpha with a combined rate of 0.85 nmol/min per nmol of cytochrome P-450 at saturating E2. These results suggest that depletion of E2 by enhanced metabolism accounts for the antiestrogenic activity of TCDD in MCF-7 cells.

Aryl Hydrocarbon Hydroxylases↗
PubMed1990

Plasma cortisol is increased during the inhibition of LH secretion by central LHRH in the ewe.

The delayed and sustained suppression of LH secretion induced by the administration of LHRH into the third cerebral ventricle of the ovariectomized ewe suggests the activation of a neuroendocrine mechanism involving components separate from the LHRH system. Endogenous opioid peptides are involved in regulating LHRH secretion but our recent work shows they do not mediate this inhibition. There is, however, clear evidence for a role for each of the components of the hypothalamopituitary-adrenal axis in the suppression of LHRH/LH secretion. Thus, the relationship between the central administration of LHRH, changes in plasma cortisol and LH secretion was investigated. Injection of LHRH (21 pmol) into the third cerebral ventricle of ovariectomized ewes caused a significant and rapid rise in plasma cortisol to a maximum of 4-5 times pre-injection values, followed by a delayed but sustained reduction in LH secretion. There was a high correlation (r = -0.902) between the increase in cortisol and the reduction in LH. Both the stimulatory effect of central LHRH on plasma cortisol and the inhibitory effect on plasma LH were blocked by prior central treatment with an LHRH antagonist. Intravenous infusion of cortisol, to reach levels observed after central LHRH administration, reduced LH secretion (although not to the levels which followed central LHRH) due in part to a reduction in pituitary responsiveness to LHRH. These experiments provide evidence that cortisol, either alone or in combination with another component(s) of the hypothalamopituitary-adrenal axis, may play a role in the LHRH-induced inhibition of LHRH/LH secretion in the sheep.

Animals↗
PubMed1990

Central administration of corticotrophin-releasing factor in the sheep: effects on secretion of gonadotrophins, prolactin and cortisol.

Stress interferes with the normal secretion of LH and FSH from the anterior pituitary gland and therefore exerts a deleterious effect on reproductive function. Evidence suggests that the stress-induced disruption of gonadal function is due to a central action of corticotrophin-releasing factor (CRF) to inhibit the release of LHRH into the hypophysial-portal circulation. The following studies were undertaken to investigate further the role of CRF in regulating gonadotrophin release in the sheep and to determine whether central administration of this peptide can alter the secretion of other hormones (e.g. prolactin and cortisol) known to be released under conditions of stress. In contrast to other species, injection of CRF into the third ventricle of the sheep brain caused a dose-related stimulation of LH secretion. The pulse frequency and mean levels of LH were increased significantly following central administration of CRF. In contrast to this effect, central administration of CRF did not alter the plasma concentration of FSH but caused a marked and dose-related stimulation of prolactin and cortisol secretion. The stimulatory effect of CRF on prolactin secretion was reversed by i.v. administration of the opioid antagonist naloxone, suggesting that endogenous opioid peptides mediate the central effect of CRF on the release of prolactin, but not cortisol. In conclusion, these data demonstrate that administration of CRF causes a dose-related stimulation of LH and prolactin release from the anterior pituitary gland and cortisol from the adrenal gland. In the case of prolactin, endogenous opioid peptides are likely to mediate this response.(ABSTRACT TRUNCATED AT 250 WORDS)

Adrenal Glands↗
PubMed1989

Inhibitory effect of central LHRH on LH secretion in the ovariectomized ewe.

The role of central luteinizing hormone releasing hormone (LHRH) in the control of pulsatile LHRH and luteinizing hormone (LH) secretion was investigated in ovariectomized adult ewes. Injection of LHRH (2.1-21 pmol) into the third cerebral ventricle caused a delayed but sustained inhibition of LH secretion. Pulse frequency, pulse amplitude and mean LH levels were reduced significantly when compared with the responses to the control injection of saline (50 microliters). The inhibitory effect of centrally administered LHRH was not accompanied by a reduction in the pituitary responsiveness to intravenous LHRH. In contrast to the effect on LH, plasma levels of follicle-stimulating hormone (FSH) and prolactin were unaffected by central LHRH. The inhibitory action of LHRH was antagonized by prior injection of an LHRH antagonist ([N-Ac-D-Nal(2)1, D-p-Cl-Phe2, D-Trp3, D-hArg (Et2)6, D-Ala10] LHRH, 69 pmol) into the third ventricle. Central injection of the LHRH antagonist alone (at the same concentration) did not influence any characteristic of pulsatile LH secretion. In conclusion, these data indicate that exogenous administration of LHRH into the brain exerts a dose-related and receptor-mediated inhibition of LHRH pulse generator activity. However, the physiological significance of endogenous LHRH in the regulation of the LHRH pulse generator remains unresolved.

Animals↗
PubMed1988

Enhancement of 2- and 16 alpha-estradiol hydroxylation in MCF-7 human breast cancer cells by 2,3,7,8-tetrachlorodibenzo-P-dioxin.

2,3,7,8-Tetrachlorodibenzo-p-dioxin exhibits antiestrogenic activity and induces cytochromes P-450 in estrogen-dependent MCF-7 human breast-cancer cells. To determine whether induction of 2- or 16 alpha-hydroxylation of 17 beta-estradiol has a role in this antiestrogenic activity, MCF-7 cells which were exposed to this xenobiotic for 72 hrs were incubated with either [2-3H] or [16 alpha-3H] 17 beta-estradiol and the extent of tritiated H2O formation, indicative of site-specific hydroxylation, was determined. 2,3,7,8-Tetrachlorodibenzo-p-dioxin-treated MCF-7 cultures showed an 8-fold increase in 2-hydroxylation and a 2-fold increase in 16 alpha-hydroxylation. These results support the suggestion that increased hydroxylation of 17 beta-estradiol may have a role in the antiestrogenic activity of 2,3,7,8-tetrachlorodibenzo-p-dioxin in MCF-7 cells.

Aryl Hydrocarbon Hydroxylases↗
PubMed1988

Inhibition of postconfluent focus production in cultures of MCF-7 human breast cancer cells by 2,3,7,8-tetrachlorodibenzo-p-dioxin.

2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), a potent inducer of differentiation and an antiestrogen, is shown to suppress in vitro postconfluent cell accumulation in the estrogen-dependent MCF-7 human breast tumor cell line. This dose-responsive suppression is apparent by 14 days of exposure with an EC50 between 10(-10) and 10(-11) M TCDD, and is characterized by reduced cell density (approximately 60% of controls after 14 days). This was attributed to a reduced formation in TCDD-treated cultures of multicellular foci which are characteristic of cancer cell growth in vitro (less than 1/mm2 compared to control levels of 40/mm2). Preconfluent cell growth and viability of MCF-7 cells is not affected by 10(-9) M TCDD. These results suggest that the principle of TCDD's activity may be useful in the study and possibly the management of estrogen-dependent breast tumors.

Breast Neoplasms↗
PubMed1988

Naloxone excites oxytocin neurones in the supraoptic nucleus of lactating rats after chronic morphine treatment.

1. Lactating rats were implanted with a cannula in a lateral cerebral ventricle to deliver morphine (up to 50 micrograms/h) chronically from a subcutaneous osmotically driven mini-pump. After infusion of morphine for 5 days the rats were anaesthetized with urethane and prepared with ventral surgery for recording the electrical activity of single, antidromically identified neurones in the supraoptic nucleus. 2. A single I.V. injection of naloxone (5 mg/kg) in these rats provoked a long-lasting, large increase in intramammary pressure, but in control rats had negligible effects. Concentrations in plasma of oxytocin, measured by radioimmunoassay in samples of femoral arterial blood, rose from 44.7 +/- 2.5 to 1072.1 +/- 89.5 pg/ml (means +/- S.E.M.) 6 min after naloxone in the morphine-treated rats. In control rats, the concentration of oxytocin in plasma rose only from 42.1 +/- 2.9 to 125.1 +/- 28.2 pg/ml after naloxone. 3. Naloxone produced a transient increase in arterial blood pressure in morphine-treated but not control rats. Concentrations in plasma of vasopressin, measured by radioimmunoassay in samples of femoral arterial blood, rose in morphine-treated rats from 7.4 +/- 2.4 to 29.2 +/- 3.7 pg/ml after naloxone, but did not rise significantly in control rats. 4. Naloxone (1-5 mg/kg) produced a prompt and prolonged increase in the discharge rate of each of ten continuously active (putative oxytocin) cells recorded from ten morphine-treated rats. The discharge rate of the six cells tested at the highest dose (5 mg/kg) increased by an average of 6.3 Hz (360%) within 5 min, and the firing rate remained elevated for at least 30 min; the discharge rate of six continuously active supraoptic neurones recorded in control rats was not affected by naloxone. 5. The firing activity of five phasic (putative vasopressin) supraoptic neurones in morphine-treated rats was increased for at least 30 min by the injection of naloxone; these increases were the result of a raised intraburst firing rate with no change in burst duration or frequency. One phasic neurone was inhibited for 15 min, and one phasic neurone was unaffected. 6. The excitatory effects of naloxone on neurones in the supraoptic nucleus of morphine-treated rats were not explained by changes in blood pressure or osmolarity and did not depend on suckling or a cholinergic pathway. 7. The concentrations of oxytocin in plasma and the operation of the milk-ejection reflex were similar in the controls and morphine-treated rats, prior to naloxone. These findings indicate tolerance to initial inhibitory effects of morphine on oxytocin secretion.(ABSTRACT TRUNCATED AT 400 WORDS)

Action Potentials↗
PubMed1988

Functional expression of rat pituitary gonadotrophin-releasing hormone receptors in Xenopus oocytes.

Expression of receptors for the hypothalamic regulatory peptide, gonadotrophin-releasing hormone (GnRH), was investigated by intracellular recording from Xenopus oocytes injected with poly(A)+ mRNA isolated from rat anterior pituitary glands. Membrane depolarizations were induced in oocytes in a dose-dependent fashion following the application of GnRH (10nM - 1 microM) or a GnRH superactive agonist, buserelin (1nM - 1 microM). The response was reversibly blocked by the addition of a GnRH antagonist (1 microM). TRH (10nM - 1 microM) had no effect on most of these oocytes. In contrast, some other oocytes which showed no responses to GnRH or to the GnRH agonist, displayed depolarizing responses to TRH (10nM - 1 microM). A relatively small number of oocytes responded to both ligands. Control oocytes did not respond to the GnRH analogues or to TRH. This successful expression of the GnRH receptor could provide a new approach to the study of the receptor, and serve as a means for the isolation and cloning of the encoding genes.

Animals↗
PubMed1987

Suppression of estrogen-regulated extracellular tissue plasminogen activator activity of MCF-7 cells by 2,3,7,8-tetrachlorodibenzo-p-dioxin.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) suppresses the estrogen enhancement of tissue plasminogen activator (t-PA) by MCF-7 breast cancer cells. 17 beta-estradiol treatment of MCF-7 cells was previously shown to enhance t-PA secretion in a receptor-mediated process dependent on RNA and protein synthesis. The current studies demonstrate that treatment with TCDD, at a concentration as low as 10(-11) M, reduces the 17 beta-estradiol-induced enhancement of t-PA secretion in these cells. Treatment of MCF-7 cells with TCDD alone does not alter t-PA activity nor was inhibition of t-PA activity observed when TCDD was added directly to the enzyme assay. Kinetic studies and the lack of inhibition following in vitro mixing of conditioned media from TCDD-treated and control 17 beta-estradiol stimulated MCF-7 cells argue against TCDD induction of a plasminogen activator inhibitor. The related polychlorinated dibenzofuran, 2,3,7,8,-tetrachlorodibenzofuran, while also active, is less potent that TCDD. Other polychlorinated dibenzodioxins, polychlorinated dibenzofurans, and polychlorinated biphenyls do not suppress 17 beta-estradiol induction of t-PA over the concentrations tested. These results are in agreement with the structure-activity relationships established using these compounds in other assay systems. Treatment with TCDD does not alter the number or affinity of 17 beta-estradiol receptors of MCF-7 cells. TCDD treatment does not suppress constitutive t-PA activity in the estrogen independent breast cancer line MDA-MB-231 nor the t-PA induced by 12-O-tetradecanoylphorbol-13-acetate in HeLa cells. These effects suggest that TCDD is not acting directly on expression of the t-PA genome. Induction of aryl hydrocarbon hydroxylase by TCDD, a cytochrome P-450 regulated metabolic enzyme for which TCDD is the most potent known inducer, was observed in MCF-7 cells but not in MDA-MB-231 or HeLa cells. A plausible mechanism for the antiestrogenic activity of TCDD is based on the metabolic conversion of 17 beta-estradiol to less active derivatives by TCDD induced cytochrome P-450 metabolic enzymes.

Adenocarcinoma↗
PubMed1987

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) does not inhibit intercellular communication in Chinese hamster V79 cells.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is a ubiquitous environmental pollutant which has been shown to be both a potent teratogen and carcinogen and also to have tumor-promoting activity. We have compared TCDD with the proto-type phorbol ester tumor promoter, 12-O-tetradecanoylphorbol-13-acetate (TPA) using the metabolic cooperation assay as a measure of tumor promotional competence. Unlike TPA, TCDD was found to be ineffective in inhibiting metabolic cooperation at concentrations which elicit many of TCDD's biological responses. These results suggest that TPA and TCDD elicit tumor promotion by different pathways. We discuss these results in the context of cell-type-specific and TCDD receptor-mediated biological responses to tumor promoters.

Animals↗
PubMed1985

Stress-induced analgesia evoked by intraperitoneal injection of hypertonic saline: evidence for its occurrence in vasopressin deficient rats.

Experiments were carried out to investigate whether vasopressin is involved in stress-induced analgesia. Intraperitoneal injection of hypertonic saline caused a significant and dose-related increase in the latency to the tail-flick response of the rat to noxious heat and was used as a stimulus for stress-induced analgesia. Neither the pituitary nor opioid peptides appeared to be involved, since the response occurred in hypophysectomized rats and was not reduced by the opiate antagonist naloxone. Furthermore hypertonic-saline analgesia was clearly potentiated in hypophysectomized rats in comparison to sham-operated controls. Hypertonic-saline analgesia was also observed in vasopressin-deficient (homozygous Brattleboro) rats similar in both magnitude and duration to that in normal rats of the same strain (Long Evans). It was concluded that vasopressin was not involved in stress-induced analgesia evoked by hypertonic saline.

Animals↗
PubMed1984

The two-dimensional polypeptide profile of terminally non-dividing human diploid cells.

We have used high resolution two-dimensional polyacrylamide gel electrophoresis (PAGE) to compare the polypeptides synthesized by normal human diploid fibroblasts cells which have significant proliferative capacity with those of cells which are terminally non-dividing. Normal cells that are terminally non-dividing (i.e., have no remaining proliferative potential) synthesize two polypeptides that are not detected in young cells which are either actively proliferating or growth arrested by incubation in medium containing a low concentration of serum (0.3%) for 72 h. Continued maintenance of young cells in the growth-arrested state ultimately leads to the detectable synthesis of one of the polypeptides synthesized by terminally non-dividing cells. Some preliminary biochemical properties of these two polypeptides are examined.

Cell Cycle↗